This project continues studies of how lipooligosaccharide (LOS) mimicry of human glycosphingolipids (GSL) enables the transmission of Neisseria gonorrhoeae in order to find ways to prevent it. LOS are outer membrane glycolipids that have a glycose moiety that consists of proximal Basal Region and three short distal chains, termed alpha beta and gamma. Many alpha chain oligosaccharides are structurally identical to those of lacto- (Lac-R), globo- (Pk (Gb3) and P1), paraglobo- (lacto-N- neotetraose (LNnT)), and gangliosyl (Ga1NAcbeta1 yields 3LNnT) series GSL. LOS are involved in attachment to and invasion of epithelial cells and in evasion of immune clearance mechanisms. Gonococci shed during gonorrhoea make larger LOS. The higher Mr LOS made by MS11mkC - a strain used in human experimentation -have polylactosamine structures. Polylactosaminylation explains the higher Mr molecules of this variant, but not those of others. Some serum resistant (serr) gonococcal strains extend the LOS beta chain to form an alpha-lactose that is parallel to the beta-lactose of the alpha chain, and meningococci can extend the gamma chain. We will structure higher Mr LOS made by clinical isolates; LOS made by serr strains, and LOS that appear to have higher order (Gb4 and P1) globosyl oligosaccharides that are isobaric (same Mr) with paraglobosyl and gangliosyl LOS, respectively. We particularly want to know whether higher Mr LOS have parallel GSL-like antennae that could cross-link epithelial cell receptors. We will continue to rely on mass spectrometric techniques. We know little about gonococcal LOS lipoidal moieties. This information is needed because the lipoidal moiety influences the conformation of the glycose moiety in ways that affects the latter's ability to bind glycoproteins, including antibodies. Available structural information from degraded LOS leaves known O-acyl lipoidal moiety heterogeneity unexplored. We will develop methods that allow us to structure intact LOS without prior degradation. MAbs have been used extensively in studies of gonococcal pathogenesis as surrogates for glycose structures; however, we do not have mAbs that discriminate among known glycose structures, much less for those that have yet to be found. We want to expand our library of mAbs to include additional specificities. These mAbs will be necessary for complete studies of the role of LOS in pathogenesis.